Many diseases and pathological conditions involve the inappropriate expression or undesirable activity of one or more cellular genes. Examples include cancer, in which one or more cellular oncogenes become activated and results in the unchecked progression of cell cycle processes. Furthermore, many human genetic diseases, such as Huntington's disease and certain prion conditions, result from the inappropriate activity of a polypeptide as opposed to the loss of its function. Drug therapy strategies for these disorders have frequently employed molecular antagonists which target the polypeptide product of the disease gene(s). Additionally, infectious diseases such as HIV have been successfully treated with molecular antagonists targeted to specific essential retroviral gene(s). The discovery or rational design of such disease gene or pathogen gene antagonists is often difficult and time-consuming. Furthermore, such molecular antagonists frequently have unanticipated effects on other cellular activities which were not targeted for therapeutic intervention. Therefore a systematic means for degrading one or more target polypeptides which cause or contribute to a disease or condition would allow the rapid development of suitable antagonist therapeutic agents once a disease-causing polypeptide or pathogen gene function is identified.
The ability to systematically antagonize specific target polypeptide activities would further allow for the general elucidation and control of the essential polypeptide-mediated functions of a host cell. Indeed, the field of genetics is essentially an approach to understanding biological processes through the systematic elimination of cellular polypeptide functions. Historically, the genetic approach has involved the development of “screens” for mutations in genes which affect a specific phenotypic trait of an organism. The great advantage of this approach has been that no prior knowledge of the molecular nature of the genes involved is required because the “screen” identifies the affected genes by marking them with mutations. The mutation involved is frequently a change in the gene's sequence which results in a loss-of-function of the encoded gene product. Unfortunately the genetic approach has many limitations. Indeed the study of essential genes, required for cell viability, is exceedingly difficult using a purely genetic approach. The implementation of “reverse genetics” in yeast (see e.g. Winston et al. (1983) Methods Enzymol 101: 211–28) and, later, in mammals (see e.g. Capecchi (1989) Science 244: 1288–92), has allowed the positive identification of a gene as essential through the inability to recover viable yeast haploid gene “knockout” spores or homozygous recessive “knockout” mice. Nevertheless, the exact biological processes in which the essential gene is involved are difficult to determine due to the inability to isolate and/or study the doomed knockout yeast spore or the inviable homozygous mouse zygote. Thus the downstream effects on specific aspects of cell function following removal of the essential gene product cannot be readily determined using these traditional “knockout” studies. Furthermore, while traditional gene “knockout” experiments may be useful in demonstrating that a given gene is essential for the life of the organism, they provide neither a convenient method for determining how rapidly cellular function ceases nor what cellular processes immediately follow the removal of the gene product, nor further do they allow for the facile determination of the rate at which so-called “second-site suppressing” mutations can arise which restore cell viability following the removal of the essential gene. These considerations are important in the selection of targets for the rational design of, for example, antibiotic or chemotherapeutic pharmaceutical agents.
The functional characterization of a specific cellular protein often relies on experimentally altering the levels of the protein of interest, and studying the biological consequences of this manipulation. Gene knockout, ribozyme, anti-sense or RNA-mediated interference (RNAI) technologies are frequently used in different eukaryotic organisms to either eliminate or reduce the levels of a cellular protein. Antisense oligonucleotides and designer ribozymes provide a means for the systematic removal of a specific gene product without the prior bioengineering of the cellular target. (see, e.g. Hargest and Williamson (1996) Gene Ther. 3: 97–102; Lipkowitz et al. (1996) Am J Kidney Dis 28: 475–92; and Bennett and Schwartz (1995) Circulation 92: 1981–93); however the successful employment of such nucleic acid antagonists has met with many difficulties. Furthermore, where the immediate removal of existing gene product is desired, antisense and ribozyme gene antagonists are relatively inefficient since existing gene products are removed only at the rate of their natural turnover.
A systematic, rational means for antagonizing the function of a specific cellular or pathogen gene would provide a powerful strategy for dealing with certain disease conditions as well as providing a means to determine and control the essential biological functions of a host cell.
A number of methods for the directed inactivation of a specific target polypeptide functions in a host eucaryotic cell have been developed. For example, in an attempt to provide for a systematic means of deriving temperature-sensitive conditional alleles of a given gene target, Dohmen et al. have devised a temperature-sensitive “degron” cassette that can be appended to any gene of interest and used to render it thermosensitive (Dohmen et al. (1994) Science 263: 1273–6). The generality with which this thermosensitive degron can be successfully applied to any target polypeptide has yet to be determined and the necessity of relying upon thermal induction for the resulting system is a major drawback. Indeed, eucaryotic cells experience a transient heat-shock response which can have profound effects on some cellular processes such as transcription. Furthermore, the requirement for induction by heat shock precludes useful application to mammalian transgenic animal systems. Another example of a system for the controlled removal of a specific gene product is the use of the ubiquitin N-end rule proteolytic system to rapidly and inducibly degrade a specific target which has been engineered to carry a predisposing non-methionine amino terminal amino acid (Moqtaderi, et al. (1996) Nature 383: 188–91). This system can be made inducible by engineering the host cell to express a key component(s) of the N-end rule proteolytic system under the control of a suitable promoter, such as a copper-inducible promoter. Still other systems have been developed for the controlled removal of a specific host gene. Notably the Cre/lox system (see e.g. Sauer (1998) Methods 14: 381–92) allows for the inducible deletion of a specific target gene through the action of the Cre site-specific DNA recombinase. Using this system, genetic switches can be designed to ablate a specific target gene in a specific tissue and at a specific time during development. One shortcoming of this method is that, following recombinational deletion of the targeted gene from the chromosome, the remaining mRNA and polypeptide products of the gene may only slowly be titrated out of the host cell through consecutive mitotic cell divisions and/or the eventual turnover of the mRNA and polypeptide by cellular ribonucleases and proteases. Thus it would be desirable to have a more rapid means for directly inactivating specific target genes in a host eucaryotic cell. A principal disadvantage of all of these systems is that they require the specific bioengineering of the target cell or host to potentiate the removal of the targeted gene function. In particular, these methods require the molecular modification of the target polypeptide-encoding gene and so it is difficult to employ them in the alteration of endogenous cellular protein levels. Therefore these techniques, while potentially useful in the identification and study of gene function in a model organism, are unlikely to be applicable to the therapeutic treatment of specific human disease conditions.
The steady state level of a cellular protein is reached when the balance is achieved between the rates of its synthesis and degradation. Altering the amount of a cellular protein has been a fundamental approach in understanding its normal cellular function. Many important technologies have been developed to manipulate cellular protein levels. Gene transfer and transgenic technologies in tissue culture or in animals are frequently used to overproduce proteins, whereas gene knockout, ribozyme or RNAI have been used to either eliminate or reduce expression of specific proteins. Although complete elimination of a cellular protein often allows the determination of the cellular functions of a protein, there are instances where the reduction in the level of a specific gene product may be desirable in order to assess the function of a specific protein in a specific process. Furthermore, complete gene knockout is sometimes not desirable, in situations in which the deletion of a particular gene has pleiotropic effects that may overshadow specific cellular functions.
Ubiquitin dependent proteolysis is a major catabolic pathway utilized by eukaryotic cells for the degradation of cellular proteins. Protein ubiquitination is catalyzed by the concerted actions of three classes of enzymes; the E1 ubiquitin-activating enzymes, the E2 ubiquitin-conjugating enzymes, and the E3 ubiquitin protein ligases (reviewed in Hochstrasser (1996) Annu Rev. Genet 30: 405–39). While E1 and E2 are primarily involved in the activation and transfer of ubiquitin, the substrate specificity of the ubiquitin pathway is conferred by the E3 ubiquitin protein ligases. Recent studies have revealed distinct E3 components and ubiquitination machineries that operate at various cellular processes to target distinct substrates for degradation.
For example, the SCF (Skp1, Cullin and F-box-containing proteins) ubiquitin protein ligases function to promote cell cycle transitions by targeting multiple cell cycle regulators for ubiquitin-dependent proteolysis (reviewed in King et al. (1996) Science 274: 1652–9; Patton et al. (1998) Genes Dev 12: 692–705; Koepp et al. (1999) Cell 97: 431–4; and references therein). Among the subunits of the SCF complexes, Skp1 and cullin are thought to form a stable core complex that is shared by different F-box-containing proteins, and interacts with E2 through the cullin subunit. Multiple F-box proteins exist that serve as receptors for recognition and recruitment of various target polypeptides to the core SCF for ubiquitination. Different F-box proteins share the common F-box domain for Skp1 binding, but utilize additional modular protein—protein interaction domains, such as WD40 or leucine-rich repeats (LRR) for binding distinct classes of substrates. Recently, a fourth subunit of SCFs, Rbx-1/Roc1, was identified that interacts with F-box proteins, and is thought to stimulate interaction between the Cdc34 ubiquitin-conjugating enzyme and SCF ubiquitin-protein ligases (Kamura et al. (1999) Science 284: 657–61; Skowyra (1999) Science 284: 662–5; Ohta et al. (1999) Mol Cell 3: 535–41; Tan et al. (1999) Mol Cell 3: 527–33). Our recent studies indicated that the yeast F-box-containing proteins, such as Cdc4p, is itself a target of the SCF-mediated ubiquitination and degradation (Zhou and Howley (1998) Mol Cell 2: 571–80). This suggests that the intact SCF complexes may be transient in nature, and that the proteolysis of the F-box proteins is essential for maintaining the SCF proteolytic activities by permitting the exchange of the substrate recognition components.
Transit through the G1/S boundary and initiation of DNA synthesis in the yeast Saccharomyces cerevisiae require ubiquitin-dependent proteolysis of the CDK inhibitor Sic1p by an SCF ubiquitin ligase (reviewed in (Deshaies (1997) Curr Opin Genet Dev 7: 7–16; King et al. (1996) Science 274: 1652–9; and references therein). Genetic analyses have identified four proteins, Cdc34p, Cdc4p, Cdc53p, and Skp1p, whose functions are required for Sic1p ubiquitination (Bai et al. (1996) Cell 86: 263–74; Mathias et al. (1996) Mol Cell Biol 16: 6634–43; Schwob et al. (1994) Cell 79: 233–44). Recently, the Sic1p ubiquitination pathway was biochemically defined by in vitro reconstitution using recombinant proteins (Feldman et al. (1997) Cell 91: 221–30; Skowyra et al. (1997) Cell 91: 209–19). The E3 activity is conferred by a multiprotein complex, designated SCFCdc4p, consisting of Skp1p, Cdc53p and Cdc4p. In association with the Cdc34p ubiquitin conjugating, enzyme, SCFCdc4p promotes Sic1p ubiquitination in vitro in the presence of the E1 ubiquitin activating enzyme and ubiquitin. Importantly, phosphorylation of Sic1p by Cdc28p/G1 cyclin dependent kinase is a prerequisite for Sic1p recognition by Cdc4p (Feldman et al. (1997) Cell 91: 221–30; Schneider et al. (1996) Science 272: 560–2; Schwob et al. (1994) Cell 79: 233–44; Skowyra et al. (1997) Cell 91: 209–19; Verma et al. (1997) Science 278: 455–60). Although the in vitro reconstitution experiments have defined the essential ubiquitin enzyme components and the biochemical mechanisms for the Cdc34p/SCFCdc4p ubiquitination pathway, the question remains as to how the individual components collaborate in vivo to deliver ubiquitin to SCFCdc4p substrates, and how the activity of the SCFcdc4p ubiquitin protein ligase complex is controlled through the various cell cycle transitions.
Mutations of the Cdc34p/SCFCdc4p pathway components affect the stability of several regulators of cell growth in addition to Sic1p, including the Cdk inhibitor Far1p (Henchoz et al. (1997) Genes Dev 11: 3046–60; McKinney et al. (1993) Genes Dev7: 833–43), the p58ctF13 subunit of the Cbf3 kinetochore assembly complex (Kaplan et al. (1997) Cell 91: 491–500), the DNA replication protein Cdc6p (Drury et al. (1997) EMBO J 16: 5966–76; Piatti et al. (1996) Genes Dev 10: 1516–31), and the GCN4p transcription factor (Kornitzer et al. (1994) EMBO J 13: 6021–30). The Cdc34p/SCFCdc4p proteolytic activity has also been implicated in phases of cell cycle other than G1/S (King et al. (1996) Science 274: 1652–9), as deletion of SIC1 allows cdc4ts, cdc34ts, cdc53ts and skp1ts cells to pass through the G1/S boundary, but they become arrested at G2-M (Bai et al. (1996) Cell 86: 263–74; Schwob et al. (1994) Cell 79: 233–44). Therefore, the Cdc34p/SCFCdc4p pathway must be active at different stages of the cell cycle to mediate the degradation of a variety of substrates (Deshaies (1997) Curr Opin Genet Dev 7: 7–16; King et al. (1996) Science 274: 1652–9).
Two additional SCF complexes, SCFGrr1p and SCFMet30p, have also been identified in S. cerevisiae and are implicated in the degradation of G1-specific cyclins or regulators of sulfur metabolism, respectively (Patton et al. (1998) Genes Dev 12: 692–705; Skowyra et al. (1997) Cell 91: 209–19). The three SCFs share in common the Cdc53p and Skp1p components, and differ in the F-box-containing subunits, Cdc4p or Grr1p or Met30p. It is these different F-box-containing proteins that serve as the substrate recognition subunits. It is currently unclear whether the individual intact SCFs are somehow regulated within cells, or whether there is an equilibrium involving, the transient assembly of SCFs with the individual F-box subunit recognition component. Here we report that the F-box-containing subunits of SCFs are intrinsically short-lived proteins. The SCFCdc4p machinery mediates the autoubiquitination and degradation of the F-box-containing Cdc4p, the substrate recognition component of SCFCdc4p. Our studies suggest a novel mechanism for the rapid assembly and disassembly of distinct SCF complexes through auto-ubiquitination of the F-box-containing SCF components, which allows the cell to respond in a timely manner to different cell cycle or environmental signals to target a variety of specific cellular proteins for proteolysis.
The SCF ubiquitin ligases are only one group of known ubiquitin ligases. Other groups of structurally related ubiquitin ligases include the HECT domain ubiquitin ligases which include mammalian E6-AP and Nedd-4, and yeast RSP5 (see Huibegtse et al. (1995) Proc Natl Acad Sci USA 92: 2563–7; Wang et al. (1999) Mol Cell Biol 19: 342–52) as well as the more recently discovered HECT domain ubiquitin ligase proteins such as mammalian Smurfl (Zhu, et al. (1999) Nature 400: 687–93), yeast TOM1 (saleh et al. (1998) J Mol biol 282: 933–46; Utsugi et al. (1999) Gene 234: 285–95), and human EDD (Callaghan et al. (1999) Oncogene 17: 3479–91). The HECT domain ubiquitin ligases effect ubiquitin conjugation, and thereby activate targeted proteolysis, of a number of polypeptide targets such as the Schizosaccharomyces pombe mitotic activating tyrosine phosphatase cdc25p (Nefsky and Beach (1996) EMBO J 15: 1301–12) and the SMADs, a group of proteins which control embryonic development and a wide variety of cellular responses to TGF-beta signals (Zhu et al. (1999) Nature 400: 687–93). While many targets of HECT-mediated ubiquitin-dependent proteolysis interact directly with their target protein, others interact through a third “adaptor” polypeptide which itself is not targeted for ubiquitination. For example, Grb10 remains unubiquitinated following its interaction with Nedd4 HECT ubiquitin ligase, and may serve to target other proteins, such as tyrosine kinase receptors, for ubiquitination (Morrione et al. (1999) J Biol Chem 274L 24094–9).
A third group of ubiquitin protein ligases are those involved in the degradation of polypeptides with particular amino-terminal amino acid residues. These include the yeast and mammalian Ubr1p N-end rule ubiquitin ligases which bind to destabilizing target polypeptide N-terminal residues and facilitate ubiquitination of the bound target (Kwon et al. (1998) Proc Natl Acad Sci USA 95: 7893–903; Varshavsky (1996) Proc Natl Acad Sci USA 93: 12142–9). This group of ubiquitin protein ligases likely function to control the intracellular levels of polypeptides which have undergone an endoproteolytic processing event which removes the normal stabilizing amino-terminal methionine residue. Particular classes of amino acid residues such as the basic residues (arginine, lysine and histidine) or the bulky hydrophobic residues (phenylalanine, leucine, tryptophan, tyrosine, and isoleucine), when exposed at the amino-terminus of the resulting processed protein, result in requitment of Ubr1p-type ubiquitin protein ligases which effect ubiquitination of the target, resulting eventually in the proteasome-dependent destruction of the target polypeptide.
It is an object of the instant invention to exploit the ubiquitin protein ligases for experimental and therapeutic purposes by providing modified ubiquitin protein ligase polypeptides which recruit target polypeptides for ubiquitin conjugation and ubiquitin-dependent proteolytic degradation by a cellular proteasome. Such modified ubiquitin protein ligases allow for the specific and regulated destruction of any target polypeptide which is cloned or for which an interaction domain is available. It is a further an object of the invention to provide inhibitors of ubiquitin protein ligases which prevent their interaction and/or ubiquitination of a target protein which is present in a host cell. These ubiquitin protein ligase inhibitors allow for the specific blocking of ubiquitin-mediated degradation of a target protein and thereby stabilize the level of the target protein in the host cell. Therefore, the invention generally provides methods and compositions to stimulate a ubiquitin protein ligase activity on a target polypeptide, or to repress a ubiquitin protein ligase activity on a target polypeptide.